我们感谢拉狄克Sindelka他的帮助，或Cas Bresilla协助与青蛙养殖和胚胎的准备。这项工作是通过grant R01DE021109到HLS劳拉Jacox经费由美国国立卫生研究院资助的赫歇尔史密斯研究生奖学金在哈佛大学和F30DE022989-01通过NIDCR的F30个人奖学金补助。

1。试剂的准备<ol><li> 10X MBS：准备1升10倍的改进巴特的盐水（MBS）的解决方案17。参照表1，试剂，成分，和指令。用蒸馏水所有的解决方案。混在烧杯中，用搅拌棒，直到充分溶解。所有溶液应在室温下进行。 </li><li> 1X MBS：稀释于900ml蒸馏水百毫升10倍的MBS解决方案，使1升1X的MBS。加0.7毫升的1M的CaCl 2溶液。 </li><li> 0.1倍MBS：稀释1X MBS准备1升的0.5倍的MBS解决方案和2升0.1倍的MBS解决方案。以1升的0.1倍的MBS解决方案，加入1毫升10毫克/毫升庆大霉素溶液。与庆大霉素的0.1倍的MBS解决方案将用于长期的胚胎培养。 </li><li>聚蔗糖/ MBS：添加15克聚蔗糖400至500ml 0.5倍的MBS解决方案。充分混合。添加搅拌棒和搅拌，直到聚蔗糖完全溶解（几个小时）。 </li><li> 70％乙醇：用稀释的100％乙醇70％乙醇蒸馏水。 </li></ol> 2。玻璃的准备工作工具<ol><li>针制剂：加载毛细管进针车夫。 <ol><li>拉将根据表2所示的设置的织针：针拔出器的设置。该设置是一个萨特仪器有限公司型号P-80/PC微量拉马和毛细管管材，作为特定的试剂和设备的表中描述。但是，这些设置是特定于萨特针车夫，并需要进行调整，其他机器。设置可以使用一个斜坡测试来确定，具体由机床制造商。 </li><li>在筹备过程拉4-6针。针应该被打破，使得玻璃尖端的柔性，毛发状部被完全除去，这通常是2-3毫米长。尖端必须是相对刚性的，但仍然足够窄以被用作切割工具。见照片图1A理想的针。 </LI&gt; <li>存储针在培养皿用粘土下来的中心带。按每一针的轴成泥，拿着它在的地方，以保持脆弱的锐利尖端离底部和两侧。 </li></ol></li><li>对于额外的工具，获得玻璃盖玻片，一对长标准模式镊子，本生灯，和3-4的玻璃巴斯德吸液管（大小5¾英寸）。 </li><li>吸管工具：为了使吸移管工具，点燃本生灯和玻璃巴斯德吸管的尖端放置到火焰的蓝色部分 ，同时旋转它，使得该尖端熔化和孔完全密封，形成一个封闭的圆形端。圆形，密封尖稍后用于使凹陷的粘土菜，在操作过程中保持胚胎。 见图1B。 </li><li>玻璃桥：为了使玻璃桥，用一个较长的标准模式由镊子盖玻片玻璃3毫米大块仔细折断3毫米。按住一块盖玻片用吨weezers，将玻璃中的火焰，直到所有四个边缘软化和曲线向下，形成一个微小的玻璃圆顶。边缘应该不再是尖锐的。参见图1C。 </li><li>通过将它们存储在玻璃盖片的桥梁，边缘向下放入橡皮泥，衬培养皿的底部。插入的桥梁，使得桥的顶部保持在粘土表面的上方。不要用力过猛，使得桥梁断裂，并不能完全嵌入桥进入粘土，因为它可以是很难去除。灭菌用火焰或70％乙醇后，将桥可以从实验中重复使用试验。 </li></ol> 3。准备胚胎操作<ol><li> A线小60毫米塑料培养皿橡皮泥。使用之间，盘子和粘土表面用蒸馏水彻底洗涤，然后用70％的乙醇。 注：使用红色，白色或黄色，凡阿肯Plastalina橡皮泥，可以在当地被买走y或艺术品商店。黑粘土不建议，因为它释放残余物。 </li><li>用3％聚蔗糖0.5倍的MBS解决方案，填补了菜。较高浓度的盐可以防止组织离解，并且该多糖的Ficoll有助于增稠溶液，这有助于保持面中的位置。 </li><li>用火烧巴斯德吸管工具（ 见图1），以使浅2-3毫米的凹陷中的粘土，约20阶段胚胎体的深度。做20-30洼地，1-2 mm处，上的菜的每一面，所以总共有40-60洼地是。标签一侧LOF / GOF，和另一侧的野生型，通过雕刻字母与镊子的粘土。 </li></ol> 4。术前准备胚胎<ol><li>获得文化非洲爪蟾使用标准方法17胚胎。青蛙养殖的详细说明，请参见西伯等 17 <ol><li>四十八小时在实验前要想获取从雌蛙卵和体外受精执行。 </li><li>注入0.5-1纳克的封膜的GFP表达，以及任何所需的基因或反义吗啉代修饰的反义寡核苷酸（“吗啉”）在一个细胞阶段或成2细胞在2细胞期。一个细胞阶段持续约70-90分钟。注入的1-3 NL的总体积。在这里的RNA编码荧光蛋白（如GFP或RFP的RNA）的，可以注入FITC标记的吗啉代或荧光标记的葡聚糖。荧光是用于确定是否移植的组织愈合，并保持在头部重要。 </li><li>封顶的mRNA可以从使用​​mMessage mMachine SP6或T7试剂盒的线性质粒准备。吗啉代可以被设计，并通过基因Tools公司订购。所需的所希望的效果吗啉的用量必须为每一个基因18来确定。 </li><li>存储注射的胚胎在15℃下进行48小时，直到它们达到19-20级。 （F或所有后续步骤，根据非洲爪蟾由Nieuwkoop和法贝尔19的普通表阶段的胚胎。） 注意：在手术当天，受援国和捐助国的胚胎必须在对方的一个阶段的移植以最佳状态工作。然而，胚胎注射吗啉（“morphants”）有时发展比野生型更慢或控制morphant胚胎，进行必要的协调实验，把它所以无论morphant和野生型胚胎是在同一个舞台。 Morphants可能需要被维持在较高的温度下12-24小时前的程序。以增加的胚胎可以在匹配阶段发现的可能性，胚胎可以保持在不同温度。实验前二十四小时，应该划分胚胎成几盘，放morphants在18-20℃和野生型的胚胎在15-18°C。 </li></ol></li><li>在移植实验中，REM的一天从the15℃培养箱奥雅纳胚胎，并根据Nieuwkoop和法贝尔19阶段他们。如果他们是年龄小于神经胚后期（19级），让它们在室温下放置1-2小时，直到他们达到阶段19。 </li><li>屏幕的荧光显微镜下注射的胚胎。选择显示均匀，明亮荧光的实验胚胎。 </li><li>除去杂波和可能的污染物的经​​营面积近。擦拭下来的操作表面，体视显微镜和所有的工具，用70％的乙醇。 </li><li>一旦胚胎正处于阶段19，删除使用＃45分之5杜蒙钳在立体显微镜下卵黄膜。 <ol><li>除去各捐助者和东道国胚胎20-30卵黄膜。 </li><li>这是第几个脸移植实验，应该练了几个胚胎每个条件。该方法是有挑战性的，需要小心实践它可用于较大规模之前，快速和成功。一个可以工作üp到20做移植/实验。 </li></ol></li><li>移动主机的胚胎成使用塑料的操作盘刻度移液管的尖端切断，使得开口比胚胎（至少几个毫米）宽得多。要小心，避免触及胚胎气泡或水面，因为胚胎会在表面张力的爆炸。 </li><li>使用＃四十五分之五钳子插入胚胎成粘土凹陷，与胚胎中的粘土后部。轻轻关闭周围使用镊子胚胎的基础粘土，留下胚胎的前25％，头部，从抑郁症突出。 </li><li>一旦所有的主机胚胎固定在其洼地，移动到板的另一侧，并开始将供体胚胎到他们的洼地。重复固定在其孔中的胚胎的过程。 </li></ol> 5。进行面部移植手术<ol><li>割刀：作为切割刀去除捐助EAD组织，使用适当碎玻璃毛细管针（见步骤2.3和图1）。 注意：一个可以直接持有的手指之间的毛细管（这适用于用小手的人），或者可以安装在针昆虫针固定器。加载到针保持器Electrosharpened钨针17可以被用来代替毛细管针。请参阅特定的试剂和设备的表中提出销户，钨针。 </li><li>在立体显微镜下，将针入胚胎的头部到水泥腺的左侧。针应深深插入时，使得它从胚胎外的推移，通过磁头，并进入前肠。移植整个EAD，削减应该从胚胎外延伸到前肠。对于外胚层只移植，削减应该是浅和扩展只能通过外胚层。 <ol><li>从左侧轻弹针向右侧的头，穿过水泥腺的整个宽度。轻弹是重要的，因为运动给人以干净的切割。参阅图2A的技术和图2B所示的切口的一个示范的概要。水泥腺体和眼睛都为削减重要的地标。切口的顺序不会影响结果，并且可以根据用户的偏好或螺旋性而有所不同。 </li></ol></li><li>将针在上切割的起点，在水泥腺的左边框，并且向上轻弹针，直到它到达左眼的底部。这会创建一个从水泥腺的左侧边界的垂直切到左眼的底部。 </li><li>将针在水泥腺的右边界，并且向上轻弹针，直到它到达右眼的底部。这会创建一个从水泥腺的右边界的垂直切到右眼的底部。 </li><li>为了充分切除组织，弗力ķ从左眼到右眼的底部的底部针，创建一个横向切，将释放组织。切口可从胚胎的外侧延伸到前肠，包括外胚层和内胚层中的EAD植体。另外，浅切，可用于外胚层只EAD移植。一旦EAD组织被去除，应该有一个矩形孔从胚胎的外进入前肠，从水泥腺拉伸到刚好在眼睛（从上到下）所示。这个洞应该从左眼内侧边界延伸到右眼（边到边）的内部边框。参见图2BB。 </li><li>轻推针的尖切除的组织，并通过缓冲抬起它包含主机胚胎的菜的一部分。请勿将组织暴露在表面的空气，否则将被损坏。 </li><li>由宿主胚切除相同的组织，作为供体。丢弃主机EAD外植体或将其保存到插入背板t代入供体的脸，因为相互移植。 </li><li>将供体植进用＃45分之5钳造成主机孔。 </li><li>一旦供体组织的位置正确，并完全插入，小心地将玻璃桥（ 见图1和图2BC）对胚胎的脸持有的移植到位。桥的两端应插入到粘土，拿着它到位。这座桥应该轻轻地施加压力，移植的组织，使得移植在于主机头平齐，没有它伸出的头部或深的头内躺着。头部可以通过盖玻片被稍扁平，但要小心，不要损坏胚胎有太大的压力。移植应在5分钟内完成。 注：一个有经验的调查员可以为每个实验进行约二十移植，过2-3小时。在此期间，将胚胎从阶段20-22进步。完成了面部移植手术在21级或22 s不影响结果。后移植（在阶段22-26）可以做到，但更难以作为波峰 - 自由EAD中线区域变窄为颅神经嵴移动到脸上。整个移植的一致性是至关重要的。 </li></ol> 6。脸部移植手术后恢复<ol><li>治疗通常需要2-3个小时。离开胚胎在室温下不受干扰地在他们的粘土洼地与玻璃桥控股供体组织到位。 </li><li>一旦移植治好了，小心取出玻璃桥，从各地使用镊子胚胎的底部去掉泥土，并用塑料刻度移液管轻轻真空胚胎他们的洼地。 </li><li>把胚胎成适当标记的培养皿中，一半充满了干净的0.1倍MBS与庆大霉素。 </li><li>成长的胚胎在15℃或18℃下了好几天，直到他们达到在40期喂养蝌蚪阶段，W母鸡面部表型可以得分。 <ol><li>与庆大霉素的0.1倍的MBS溶液应每天更换，任何死胚应及时清除，以防止污染其他胚胎的死亡。 </li><li>口开在阶段40。在40-41阶段，必须检查移植的组织仍然未愈的地方通过查看它的荧光。移植偶尔掉下来，所以必须确保所有打进胚胎有供体组织到位。 </li></ol></li></ol>

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Early Development of Xenopus laevis. , (2000).</li><li>Tandon, P., Showell, C., Christine, K., Conlon, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morpholino+injection+in+Xenopus.">Morpholino injection in Xenopus.</a> Methods Mol. Biol. 843, 29-46 (2012).</li><li>Nieuwkoop, P. D., Faber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Normal Table of Xenopus laevis (Daudin). , (1994).</li></ol>

作者什么都没有透露。

关键步骤和限制：EAD换脸程序的时间和密集的工作。它需要实践，稳定的双手，灵巧和完善。换脸协议依赖于研究人员的能力，有效地去除和移植组织。如果时间过长插入移植到宿主的脸，主机脸会开始收缩和愈合。镊子可以用来微妙地扩大面部区域。然而，如果已经发生显著伤口收缩，移植不会愈合，以及与可能需要减小尺寸，以适应宿主的面部孔。移植的尺寸和形状必须大致相匹配的?...

要了解颅面发育过程中相关颅面先天缺陷1-2，重要组织和他们的贡献的信令机制必须进行标识。在青蛙非洲爪蟾中，面部的一部分，包括口腔和鼻孔形式从“极端前域”（EAD），其中外胚层和内胚层直接并置3-4。在EAD也作为一个信令中心来影响周围的组织，包括颅神经嵴，形成钳口和其他面部区域5。识别基因，有助于EAD功能，一个“脸移植”技术被开发，其中组织从供体移植到宿主胚胎，除去对应主机区域之后。继移植，导致面部发育评估。因此，功能丧失（LOF）或功能（GOF）中的EAD特定基因增益的效果的局部分析，其中的h时休息EAD和身体是由野生型的组织。的倒数可移植的执行，其中野生型组织被移植到与全局LOF或GOF中的胚胎特异性基因。移植已被经常使用在非洲爪蟾和小鸡研究6。例如， 非洲爪蟾移植已经解决了纯合神经诱导，镜头和神经的能力，和神经嵴迁移7-10。鹌鹑鸡嵌合体移植术分析了前神经板，前神经嵴，神经嵴和颅骨11-14的发展。这是在非洲爪蟾颅面部发育的研究第一次移植技术，该技术已经证明了的Wnt抑制剂Frzb1和红新月会在推定口5调节基底膜形成了一种新的作用。 非洲爪蟾是颅面部发育的研究的理想模型因为胚胎是大的，发展对外，一个ND面对的是随时可见，让显微和发展成像。机制相关的面部发育出现保守，表明在青蛙作出裁断洞察人类发展4,15-16。

<table border="0" cellpadding="0" cellspacing="0" width="559">
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Pasteur pipette</td>
			<td rowspan="3" style="width:101px;">VWR</td>
			<td rowspan="3" style="width:136px;">14672-400</td>
			<td style="width:128px;">Lime Glass&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Size 5 3/4&#8217;&#8217;</td>
			<td style="width:128px;">Cotton Plugged</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;"></td>
			<td style="width:128px;">Disposable</td>
		</tr>
		<tr height="20">
			<td height="41" rowspan="2" style="height:41px;width:195px;">Graduated Transfer Pipette</td>
			<td rowspan="2" style="width:101px;">VWR</td>
			<td rowspan="2" style="width:136px;">16001-180</td>
			<td style="width:128px;">Disposable&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Polyethylene</td>
		</tr>
		<tr height="80">
			<td height="100" rowspan="2" style="height:100px;width:195px;">#5/45 forceps</td>
			<td rowspan="2" style="width:101px;">Fine Science Tools by Dupont medical</td>
			<td rowspan="2" style="width:136px;">11251-35</td>
			<td rowspan="2" style="width:128px;">Angled 45 degrees</td>
		</tr>
		<tr height="20">
		</tr>
		<tr height="40">
			<td height="81" rowspan="3" style="height:81px;width:195px;">Standard Pattern Forceps</td>
			<td rowspan="3" style="width:101px;">Fine Science Tools</td>
			<td rowspan="3" style="width:136px;">11000-20</td>
			<td style="width:128px;">Straight; serrated tip</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:128px;">Stainless Steel;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">20cm long</td>
		</tr>
		<tr height="60">
			<td height="81" rowspan="2" style="height:81px;width:195px;">Capillary Tubing (for needles)</td>
			<td rowspan="2" style="width:101px;">FHC</td>
			<td rowspan="2" style="width:136px;">30-30-1</td>
			<td style="width:128px;">Borosil 1.0mm OD x 0.5mm ID/Fiber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">100mm each</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Cover slip&#160;</td>
			<td rowspan="4" style="width:101px;">VWR</td>
			<td style="width:136px;">48393 252&#160;</td>
			<td style="width:128px;">24x60mm&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">micro cover glass</td>
			<td style="width:136px;">or&#160;</td>
			<td style="width:128px;">or&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">(for glass bridges)</td>
			<td style="width:136px;">48393 230</td>
			<td style="width:128px;">24x40mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">No.1.5</td>
			<td style="width:136px;"></td>
			<td style="width:128px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:195px;">Ficoll 400</td>
			<td style="width:101px;">Sigma-Aldrich</td>
			<td style="width:136px;">F9378</td>
			<td style="width:128px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">Needle Puller&#160;</td>
			<td style="width:101px;">Sutter Instrument Co</td>
			<td rowspan="3" style="width:136px;">Needle Puller: discontinued Filament: FB300B</td>
			<td rowspan="3" style="width:128px;">The most similar, currently available&#160; needle puller is the P-97. For filaments, use Sutter 3.00mm square box filaments, 3.0mm wide.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:195px;">Model P-80</td>
			<td style="width:101px;">Flaming / Brown micropipette puller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">(discontinued)</td>
			<td style="width:101px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Stereomicroscope</td>
			<td rowspan="2" style="width:101px;">Zeiss</td>
			<td rowspan="2" style="width:136px;"></td>
			<td rowspan="2" style="width:128px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Zeiss Stemi 1000</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:195px;">Stereomicroscope Lighting by Fostec</td>
			<td style="width:101px;">Fostec</td>
			<td style="width:136px;"></td>
			<td style="width:128px;">Use a light box with 2 fiberoptic arms.&#160;&#160;</td>
		</tr>
		<tr height="60">
			<td height="81" rowspan="2" style="height:81px;width:195px;">Nickel Plated Pin Holder</td>
			<td rowspan="2" style="width:101px;">Fine Science Tools</td>
			<td rowspan="2" style="width:136px;">26018-17</td>
			<td style="width:128px;">Jaw Opening Diameter: 0 to 1mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Length: 17cm</td>
		</tr>
		<tr height="60">
			<td height="101" rowspan="3" style="height:101px;width:195px;">Moria Nickel Plated Pin Holder</td>
			<td rowspan="3" style="width:101px;">Fine Science Tools</td>
			<td rowspan="3" style="width:136px;">26016-12</td>
			<td style="width:128px;">Jaw opening Diameter: 0 to 1mm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:128px;">Length: 12cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;"></td>
		</tr>
		<tr height="40">
			<td height="60" rowspan="2" style="height:60px;width:195px;">Tungsten Needles</td>
			<td rowspan="2" style="width:101px;">Fine Science Tools</td>
			<td rowspan="2" style="width:136px;">10130-05</td>
			<td rowspan="2" style="width:128px;">0.125mm Rod diameter</td>
		</tr>
		<tr height="20">
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Van Aken Plastalina</td>
			<td rowspan="2" style="width:101px;">Blick&#160;</td>
			<td rowspan="2" style="width:136px;">#33268-2981</td>
			<td rowspan="2" style="width:128px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:195px;">Modeling Clay- white, red or yellow</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:195px;">mMessage mMashine SP6 or T7 Kit</td>
			<td style="width:101px;">Ambion</td>
			<td style="width:136px;">AM1340</td>
			<td style="width:128px;"></td>
		</tr>
	</tbody>
</table>

facial transplants in xenopus laevis embryos

颅面先天缺陷发生在1每700出来的活产婴儿，但病因却很少，由于颅面部发育的了解有限知。以识别其中的显影脸的图案形成过程中信号转导途径和组织行动，一个“脸移植”技术已发展中的青蛙非洲爪蟾胚胎。推定面巾纸（在“至尊前域”（EAD））的区域从供体胚胎tailbud阶段取出，并移植到同一个舞台主机胚胎，从中相当于区域已被删除。这可以用于产生嵌合面的主机或供体组织具有在基因中的损耗或增益函数，和/或包括一个谱系标签。愈合后，发展的结果进行监测，并表示捐赠者或周围的宿主组织内的信号转导通路的作用。 非洲爪蟾是面对发展的宝贵的典范，作为面部区域很大，随手一ccessible的显微操作。许多胚胎可以检测，在很短的时间内开发以来迅速发生。青蛙发现是有关人的发展，因为颅面过程出现爪蟾和哺乳动物之间的保守性。

移植组织应完全插入主机头移植后， 如图3A所示，并有一个玻璃桥适当地放置在胎儿的脸， 如图2BC。移植的供体组织必须正确尺寸为主机开放，为移植成功。在EAD组织不应从头部伸出，以任何方式，如在图3B和3C所示。此外，表面移植不应转动相对于其在供体身体的位置， 如图3D。几个小时后，将移植的组织与周围面应该愈合，并且?...

Watch this Scientific Journal Video about Facial Transplants in Xenopus laevis Embryos at JoVE.com

Facial Transplants in Xenopus laevis Embryos

的技术移栽“至尊前域” 非洲爪蟾的面巾纸蟾胚胎已经研制成功。组织可以从一个基因表达的背景被转移到另一个，允许对颅面发展和信令面部区域之间的相互作用当地要求的研究。

在面部移植<em&gt;非洲爪蟾</em&gt;胚胎

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

facial-transplants-in-xenopus-laevis-embryos

Research

JoVE Journal

Biology

Facial Transplants in Xenopus laevis Embryos

10.1K 次观看.  Harvard University. 的技术移栽“至尊前域” 非洲爪蟾的面巾纸蟾胚胎已经研制成功。组织可以从一个基因表达的背景被转移到另一个，允许对颅面发展和信令面部区域之间的相互作用当地要求的研究。 

视频: Facial Transplants in Xenopus laevis Embryos

Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay

This video demonstrates the technique used for preparation of organizer and animal pole explants from Xenopus laevis embryos, including the use of the eyebrow knife - a specialized dissection tool made of one's eyebrow. The protocol for assembling an adhesion assay is also given, which probes for the presence of key adhesion molecules present on the surface organizer or animal pole cells that are critical for proper development.

组织者和动物极植从非洲爪蟾胚胎和装配一个细胞粘附实验的剖析

科研

生物学

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Plastic sections maintain true tissue morphology in thin sections of tissue that can be immunostained with fluorescent secondary antibodies, making this method more useful than paraffin-embedded or frozen sections for many types of tissue. The method for staining, plastic embedding, and sectioning is demonstrated in this video.

塑料嵌入和非洲爪蟾胚胎切片

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

从非洲爪蟾女性获得卵子

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

制备及分馏爪蟾卵提取

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

非洲爪蟾卵母细胞的微量注射

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development of a larger region of the embryo. This makes defining its role in a particular tissue difficult to analyze. For example, noggin overexpression in Xenopus laevis embryos causes the expansion of the entire anterior region, including the eye1,2. From this result, it is not known if Noggin plays a direct role in eye determination or that by causing an expansion of neural tissue, Noggin indirectly affects eye formation. Having this complex phenotype makes studying its eye-specific role in cell fate determination difficult to analyze. We have developed an assay that overcomes this problem. Taking advantage of the pluripotent nature of the Xenopus laevis animal cap 3, we have developed an assay to test the ability of gene product(s), like noggin or the eye field transcription factors (EFTFs), to transform caps into particular tissue or cell types by transplanting this tissue onto the side of the embryo 4. While we have found either Noggin protein treatment or a collection of transcription factors can determine retinal cell fate in animal caps, this procedure could be used to identify gene product(s) involved in specifying other tissues as well.

Tissue Determination Using the Animal Cap Transplant ACT Assay in Xenopus laevis

Animal caps overexpressing gene product(s) are transplanted to the flank of developing Xenopus laevis embryos in order to establish whether tissue is determined.

组织测定动物第移植（ACT）的含量非洲爪蟾</em

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

活细胞成像和胚胎上皮细胞的定量分析非洲爪蟾</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

生产转基因非洲爪蟾限制性内切酶介导的整合和核移植

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of neurulation, migrates extensively towards various organs, and differentiates into many types of derivatives (neurons, glia, cartilage and bone, pigmented and endocrine cells). In this protocol, we describe how to dissect the premigratory cranial NC from Xenopus laevis embryos, in order to study NC development in vivo and in vitro. The frog model offers many advantages to study early development; abundant batches are available, embryos develop rapidly, in vivo gain and loss of function strategies allow manipulation of gene expression prior to NC dissection in donor and/or host embryos. The NC explants can be plated on fibronectin and used for in vitro studies. They can be cultured for several days in a serum-free defined medium. We also describe how to graft NC explants back into host embryos for studying NC migration and differentiation in vivo.

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

This protocol describes how to dissect premigratory cranial neural crest (NC) from Xenopus laevis neurulas. These explants can be plated on fibronectin and cultured in vitro, or grafted back into host embryos. This technique allows studying the mechanisms of NC epithelium-to-mesenchyme transition, migration, and differentiation.

剖析<em&gt;非洲爪蟾</em&gt;神经嵴的<em&gt;在体外</em&gt;外植体培养或<em&gt;在体内</em&gt;移植

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of ...

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and widely-utilized +TIPs for analyzing MT dynamics is the End-Binding protein, EB1, which binds all growing MT plus-ends, and thus, is a marker for MT polymerization1. Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs1-3. Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker4, following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones5. This software is a MATLAB-based, open-source, user-friendly package that combines automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. Here, we present the protocol for using plusTipTracker for the analysis of fluorescently-labeled +TIP comets in cultured Xenopus laevis growth cones. However, this software can also be used to characterize MT dynamics in various cell types6-8.

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

The MATLAB-based, open source software package, plusTipTracker, can be used to analyze image series of fluorescently-labeled +TIPs to quantify microtubule dynamics.

使用plusTipTracker软件测量微管动力学<em&gt;非洲爪蟾</em&gt;生长锥

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and ...